Gene silencing is a common phenomenon in eukaryotes, whereby the expression of particular genes is reduced or even abolished through a number of different mechanisms ranging from mRNA degradation (post transcriptional silencing) over repression of protein synthesis to chromatin remodeling (transcriptional silencing).
The gene-silencing phenomenon has been quickly adopted to engineer the expression of different target molecules. Initially, two predominant methods for the modulation of gene expression in eukaryotic organisms were known, which are referred to in the art as “antisense” downregulation or “sense” downregulation.
In the last decade, it has been demonstrated that the silencing efficiency could be greatly improved both on quantitative and qualitative level using chimeric constructs encoding RNA capable of forming a double stranded RNA by basepairing between the antisense and sense RNA nucleotide sequences respectively complementary and homologous to the target sequences. Such double stranded RNA (dsRNA) is also referred to as hairpin RNA (hpRNA).
The following references describe the use of such methods:
Fire et al., 1998 (Nature 391, 806-811) describe specific genetic interference by experimental introduction of double-stranded RNA in Caenorhabditis elegans. 
WO 99/32619 provides a process of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and or a portion of the target gene are identical.
Waterhouse et al. 1998 (Proc. Natl. Acad. Sci. USA 95: 13959-13964) describe that virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and anti-sense RNA. The sense and antisense RNA may be located in one transcript that has self-complementarity.
Hamilton et al. 1998 (Plant J. 15: 737-746) describe that a transgene with repeated DNA, i.e., inverted copies of its 5′ untranslated region, causes high frequency, post-transcriptional suppression of ACC-oxidase expression in tomato.
WO 98/53083 describes constructs and methods for enhancing the inhibition of a target gene within an organism which involve inserting into the gene silencing vector an inverted repeat sequence of all or part of a polynucleotide region within the vector.
WO 99/53050 provides methods and means for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid. These molecules are capable of forming a double stranded RNA region by base-pairing between the regions with the sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprise simultaneously both sense and antisense nucleotide sequences.
WO 99/49029 relates generally to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular to a transgenic animal or plant. Synthetic genes and genetic constructs, capable of forming a dsRNA which are capable of repressing, delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.
WO 99/61631 relates to methods to alter the expression of a target gene in a plant using sense and antisense RNA fragments of the gene. The sense and antisense RNA fragments are capable of pairing and forming a double-stranded RNA molecule, thereby altering the expression of the gene. The present invention also relates to plants, their progeny and seeds thereof obtained using these methods.
WO 00/01846 provides a method of identifying DNA responsible for conferring a particular phenotype in a cell which method comprises a) constructing a cDNA or genomic library of the DNA of the cell in a suitable vector in an orientation relative to (a) promoter(s) capable of initiating transcription of the cDNA or DNA to double stranded (ds) RNA upon binding of an appropriate transcription factor to the promoter(s); b) introducing the library into one or more of cells comprising the transcription factor, and c) identifying and isolating a particular phenotype of a cell comprising the library and identifying the DNA or cDNA fragment from the library responsible for conferring the phenotype. Using this technique, it is also possible to assign function to a known DNA sequence by a) identifying homologues of the DNA sequence in a cell, b) isolating the relevant DNA homologus(s) or a fragment thereof from the cell, c) cloning the homologue or fragment thereof into an appropriate vector in an orientation relative to a suitable promoter capable of initiating transcription of dsRNA from said DNA homologue or fragment upon binding of an appropriate transcription factor to the promoter and d) introducing the vector into the cell from step a) comprising the transcription factor.
WO 00/44914 also describes composition and methods for in vivo and in vitro attenuation of gene expression using double stranded RNA, particularly in zebrafish.
WO 00/49035 discloses a method for silencing the expression of an endogenous gene in a cell, the method involving overexpressing in the cell a nucleic acid molecule of the endogenous gene and an antisense molecule including a nucleic acid molecule complementary to the nucleic acid molecule of the endogenous gene, wherein the overexpression of the nucleic acid molecule of the endogenous gene and the antisense molecule in the cell silences the expression of the endogenous gene.
Smith et al., 2000 (Nature 407: 319-320) as well as WO 99/53050 described that intron containing dsRNA further increased the efficiency of silencing. Intron containing hairpin RNA is often also referred to as ihpRNA.
Although gene silencing was initially thought of as a consequence of the introduction of aberrant RNA molecules, such as upon the introduction of transgenes (transcribed to antisense sense or double stranded RNA molecules) it has recently become clear that these phenomena are not just experimental artifacts. RNA mediated gene silencing in eukaryotes appears to play an important role in diverse biological processes, such as spatial and temporal regulation of development, heterochromatin formation and antiviral defense.
All eukaryotes possess a mechanism that generates small RNAs which are then used to regulate gene expression at the transcriptional or post-transcriptional level. Various double stranded RNA substrates are processed into small, 21-24 nucleotide long RNA molecules through the action of specific ribonucleases (Dicer or Dicer-Like (DCL) proteins). Dedicated dsRNA binding (DRB) proteins associate with DCL proteint to optimize processing of their various dsRNA substrates into specifically sized small RNAs. These small RNAs serve as guide molecules incorporated into protein complexes (RNA-induced silencing complexes (RISC)) which further contain one member of the conserved Argonaute protein (AGO) family, which lead to the various effects achieved through gene silencing. Plants such as Arabidopsis have a broad spectrum of endogenous RNA silencing pathways owing to the presence of several specialized DCL proteins (four in Arabidopsis) and distinct AGO paralogs (ten in Arabidopsis).
Small RNAs involved in repression of gene expression in eukaryotes through sequence specific interactions with RNA or DNA are generally subdivided in two classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). These classes of small RNA molecules are distinguished by the structure of their precursors and by their targets. miRNAs are cleaved from the short, imperfectly paired stem of a much larger foldback transcript and regulate the expression of transcripts to which they may have limited similarity. siRNAs arise from a long double stranded RNA (dsRNA) and typically direct the cleavage of transcripts to which they are completely complementary, including the transcript from which they are derived (Yoshikawa et al., 2005, Genes & Development, 19: 2164-2175).
The number of Dicer family members varies greatly among organisms. In humans and C. elegans there is only one Dicer. In Drosophila, Dicer-1 and Dicer-2 are both required for small interfering RNA directed mRNA cleavage, whereas Dicer-1 but not Dicer-2 is essential for microRNA directed repression (Lee et al., 2004 Cell 75:843-854, Pham et al., 2004 Cell 117: 83-94).
Plants, such as Arabidopsis, appear to have at least four Dicer-like (DCL) proteins and it has been suggested in the scientific literature that these DCLs are functionally specialized (Qi et al., 2005 Molecular Cell, 19, 421-428)
DCL1 processes miRNAs from partially double-stranded stem-loop precursor RNAs transcribed from MIR genes (Kurihara and Watanabe, 2004, Proc. Natl. Acad. Sci. USA 101: 12753-12758).
DCL3 processes endogenous repeat and intergenic-region-derived siRNAs that depend on RNA-dependent RNA polymerase 2 and is involved in the accumulation of the 24nt siRNAs implicated in DNA and histone methylation (Xie et al., 2004, PLosBiology, 2004, 2, 642-652).
DCL2 appears to function in the antiviral silencing response for some, but not all plant-viruses ((Xie et al., 2004, PLosBiology, 2004, 2, 642-652).
Several publications have ascribed a role to DCL4 in the production of trans-acting siRNAs (ta-siRNAs). ta-siRNAs are a special class of endogenous siRNAs encoded by three known families of genes, designated TAS1, TAS2 and TAS3 in Arabidopsis thaliana. The biogenesis pathway for ta-siRNAs involves site-specific cleavage of primary transcripts guided by a miRNA. The processed transcript is then converted to dsRNA through the activities of RDR6 and SGS3. DCL4 activity then catalyzes the conversion of the dsRNA into siRNA duplex formation in 21-nt increments (Xie et al. 2005, Proc. Natl. Acad. Sci. USA 102, 12984-12989; Yoshikawa et al., 2005, Genes & Development, 19: 2164-2175; Gasciolli et al., 2005 Current Biology, 15, 1494-1500). As indicated in Xie et al. 2005 (supra) whether DCL4 is necessary for transgene and antiviral silencing remains to be determined.
Dunoyer et al. 2005 (Nature Genetics, 37 (12) pp 1356 to 1360) describe that DCL4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.
Dunoyer et al. 2007 (Nature Genetics 39 pp 848-856) summarizes the different pathways in Arabidopsis as follows: DCL1, together with the DRB protein HYL1, catalyzes release of miRNAs from imperfect fold-back precursor transcripts. Generally, miRNA-loaded AGO1 then promotes cleavage of cellular transcripts carrying miRNA target sequences 8. DCL3 produces 24-nt DNA repeat-associated siRNAs that guide heterochromatin formation by recruiting AGO4 and/or AGO6. DCL4, together with DRB4, generates 21-nt trans-acting siRNAs (tasiRNAs) that require AGO1 or AGO7 functions to mediate posttranscriptional silencing of genes controlling heteroblasty and leaf polarity. Finally, DCL2 produces natural antisense transcript siRNAs orchestrating stress responses 16.
Unpublished PCT application PCT/AU07/000583 describes the modulation of and demonstrates the involvement of DCL4 in the processing of long hairpin RNA molecules into interfering RNA components that ultimately effect gene-silencing.
Although RNAi mediated gene silencing has become an accepted research tool, as well as a tool for developing particular traits, there are incidental reports raising questions on the long-term stability of gene-silencing under particular conditions, or over the life-time of several generations, particularly in plant cells and plants. E.g. Szittya et al. 2003 (EMBO Journal 22, pp 633 to 640) reported that low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. Karmeda et al. 2004 report a temperature-sensitive gene silencing by an expressed inverted repeat in Drosophila (Biochem. Biophys. Res. Comm. 315, 599-602.) Niu et al. 2006 (Nature Biotechnology 24, 1420-1428) describe that viral infection systems by TYMV symptoms on Arabidopsis plants were more severe at 15° C. than at 24° C. and that transgenic plants comprising microRNA directed to downregulate the virus expression exhibited a stable amiRNA-mediated specific virus resistance maintained at 15° C.
In addition, some applications of RNAi, particularly as a trait development tool, require a severe downregulation of the target gene, preferably even the almost complete or complete downregulation for all practical means and purposes of the target gene function.
Applying RNAi to different target genes using different chimeric genes or RNA molecules which are processed via the same siRNA molecule producing pathway may lead to a saturation of such pathways.
Accordingly, described hereinafter in the different embodiments, examples and claims are methods and means for gene silencing that address the above mentioned problems, by providing and using at least two inhibitory RNA molecules or genes encoding such RNA molecules directed to the same target nucleic acid, whereby the inhibitory RNA molecules result in gene silencing through two or more separate pathways processing the RNAi into siRNA molecules, usually mediated by different Dicer proteins or Dicer like proteins (optionally in combination with different dsRNA binding proteins and/or members of the Argonaute family) and ultimately leading to gene silencing. To alleviate the problem of saturation of RNAi processing pathways methods and means are described providing and using at least two inhibitory RNA molecules or genes encoding such RNA molecules directed to different target nucleic acids, whereby the inhibitory RNA molecules result in gene silencing through two or more separate pathways processing the RNAi into siRNA molecules.